Ca2+-calmodulin inhibits Ca2+ release mediated by type-1, -2 and -3 inositol trisphosphate receptors

InsP(3) binding to type-1, but not type-3, InsP(3) receptors is inhibited by Calmodulin in a CA(2+)-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and CA(2+) mobilization by type-1 InsP(3) receptors of cerebellum is inhibited by Calmodulin [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627-11632]. Using cell types expressing predominantly type-1, -2 or -3 InsP(3) receptors, we show that InsP(3)-evoked CA(2+) mobilization from each is similarly inhibited by Calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, Calmodulin (IC(50) approximately 15 microM) inhibited InsP(3)-evoked CA(2+) release only in the presence of CA(2+). The inhibition was unaffected by Calcineurin inhibitors. The effect of Calmodulin did not result from enhanced metabolism of InsP(3) because Calmodulin also decreased the sensitivity of the CA(2+) stores to adenophostin A, a non-metabolizable InsP(3)-receptor agonist. Protein kinase A-catalysed phosphorylation of type-1 InsP(3) receptors was unaffected by CA(2+)-calmodulin. Using a scintillation proximity assay to measure (125)I-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP(3) receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound (125)I-calmodulin. The higher-affinity site (cyt11) was also photoaffinity labelled with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)-calmodulin. We speculate that CA(2+)-independent binding of Calmodulin to a site within the first 159 residues of the type-1 InsP(3) receptor inhibits InsP(3) binding and may thereby regulate the kinetics of CA(2+) release. CA(2+)-dependent inhibition of CA(2+) release by Calmodulin is mediated by a different site: it may reside on an accessory protein that associates with all three receptor subtypes, or CA(2+)-calmodulin binding to a site lying between residues 1499 and 1649 of the type-1 receptor may inhibit CA(2+) release from any tetrameric receptor that includes a type-1 subunit.